Glycosylation deficiency phenotypes resulting from depletion of GDP-mannose pyrophosphorylase in two yeast species

• Published in: Molecular microbiology Vol. 36; no. 5; pp. 1156 - 1166
• Main Authors: Warit, S, Zhang, N, Short, A, Walmsley, R.M, Oliver, S.G, Stateva, L.I
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.06.2000; Blackwell Publishing Ltd
• Subjects: Alcian blue; Candida albicans; Candida albicans - enzymology; Candida albicans - genetics; Candida albicans - growth & development; GDP-mannose pyrophosphorylase; GDPmannose pyrophosphorylase; Glycosylation; hygromycin B; MET3 gene; Mutagenesis; Nucleotidyltransferases - genetics; Nucleotidyltransferases - physiology; Phenotype; PSA1 gene; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; SRB1 gene; Yeasts
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1046/j.1365-2958.2000.01944.x

The genes encoding GDP‐mannose pyrophosphorylase from Saccharomyces cerevisiae (SRB1/PSA1) and Candida albicans (CaSRB1) were expressed under the control of the tightly regulated promoters of MET3 and CaMET3 respectively. Northern analysis showed that the addition of methionine effectively blocks the transcription of pMET3‐SRB1/PSA1 and pCaMET3CaSRB1 expression cassettes, which had been integrated into the genomes of appropriate mutants. Methionine‐mediated repression of CaSRB1 caused loss of viability in C. albicans, demonstrating that, as in S. cerevisiae, the gene is essential for growth. Depletion of GDP‐mannose pyrophosphorylase had a highly pleiotropic effect in the two yeasts. The major phenotypes observed were lysis, failure of cell separation and/or cytokinesis, impaired bud growth and bud's site selection, clumping and flocculation, as well as increased sensitivity to a wide range of antifungal drugs and cell wall inhibitors, and impaired hyphal switching ability. These phenotypes resulted from defects in glycosylation, as demonstrated by reduced affinity for Alcian blue and sensitivity to hygromycin B. Our results provide new information about the roles of protein glycosylation in yeast and, in particular, the steps that require GDP‐mannose in the fungal pathogen C. albicans.